Make intro more relevant to this lab Improve references Images out of order Improve captions
Flow of discussion hard to follow
Missing error analysis
Title: Successfully Preparing and Characterizing a Protein Sample
Introduction: This was a three part lab that was began the week before spring break and ended just this last week.The first part involved protein expression followed by purification and then characterization. Protein expression involves the separation of the protein from the actual bacterial cells. Purification is the process used to further isolate a target protein using different scientific process and characterization involves prepping the protein samples for identification and analysis using gel electrophoresis. Recombinant DNA and protein expression has become more commercially accessible due to the methods of production becoming more cost efficient to even the youngest of aspiring scientists particularly biologists and health care scientists.Workers in the this field of research generally refer to informative websites such as the Protein Data Bank to acquire knowledge on proteins before conducting research. Different types of proteins can be analyzed using purification methods. Some of these include human proteins, bacterial proteins, fungal proteins, protozoan proteins and other types of animal proteins. Before expression can occur, the protein that is going to be used in testing needs to be acquired. One of the main ways this is done is through cloning of the various kinds of proteins. This method allows for variants of the original protein to be created usually by adding different tags or expression hosts.
Materials & Methods: The first step of the lengthy process was protein expression. In order for this to occur, a sample of E. coli bacteria was heat-shocked and spun using a centrifuge to allow access to the protein. Colonies of this protein, pGR-22, were produced over approximately twenty four hours inside of clean agar plates. LB and ampicillin were added and the final samples were harvested in Eppendorf tubes. A pellet was formed in pellet was then saved for purification. The following week, wash buffer was added and two washes were performed, purifying the protein. A NanoDrop spectrophotmeter was used to test the concentration of the protein at different wavelenght 280nM and 540 nM. In the final part of the experiment, electrophoresis was performed followed by gel drying. Before electrophoresis took place, the six samples collected from protein purication were treated with purple dye to distinguish the proteins once they were inserted into the gel. The gel was dried out in a gel dryer for one and a half hours. The finished product was compared to a molecular weight standard retrieved from the internet to determine the molecular weight and estimated purity.
Results:
Fig. 1: 15 mL conical tube showing the protein after the 2nd wash.
Fig. 2: Pellet containing the BL21(DE3) competent cells transformed with pGEM-gbr22 obtained after centrifuging for 10 min. at 4°C and rotational speed of 5,000 rpm. The weight of the pellet was approx .74g.
Fig. 3: Flask containing BL21(DE3) bacterial cells transformed using pGEM-gbr22 at 48 hrs into growth in a shaking incubator at (37˚C) and a spin setting of 250 rpm.
Fig. 4: Agar plate containing the "fun sample", human saliva after overnight incubation at 37 degrees celcius.
Fig. 6: Agar plate containing Ampicillin + BL21(DE3) cells after overnight incubation at 37˚C using no pGEM-gbr22 plasmid.
Fig. 5: Agar plate with Ampicillin + BL21(DE3) cells + pGEM-gbr22 plasmid after overnight incubation at 37 degrees celcius
Fig. 7: latter used as molecular weight standard .
Fig. 7: Absorbance vs. Wavelenght of elution 1 NanoDrop at wavelenght of 280 nm providing a yield of .21 mg/mL
Fig. 9: Elution 2 stored in a 15 mL conical tube after a second washing of the protein.
Fig.10: Gel dried in heat dryer showing protein purity from characterization.
For Beer's Law, the equation A=ebc is used. If concentration, wavelenght, and absorbance are given or found, then molar absorbance can be calculated from this formula. (.211)=(38,850)(1cm)(c) so c=(5.43*10^-6 moles per liter). The concentration is then multiplied by the molality (25794.2 grams per mol) which gives .140 grams per liter. or .140 miligrams per milliliter.
Discussion: Lysozyme is used during protein expression in order to break down the cell membranes of escherichia coli.This aids in preparing for bacterial protein extraction. Benzonase was used to digest any DNA and RNA from the bacterial cells in order to reduce the viscosity of the solution. Histadine is able to bond with Nickel (Ni) which is then able to be left behind with the column due to the size of the complex being too large to filter through with the protein. Sample 2 consists of the protein after the benzonase has been added and gone through the the centrifuge. Sample 3 is stored after the Ni-Nta is added to the protein and syringed in a filter. After this step is when the imidiazole is added that begins competing with the Histadine tags and a sample of this is taken as number 4. Sample 5 contains more of the protein than number 6 due to it being the first elution and sample 6 being elution 2. These elutions were performed by adding a highly concentrated amount of imidiazole to the solution used for sample 4. Again the main difference between the wash and elution buffers is the concentration of the imidazole involved with that of the elution buffers being much strong so as to strip away more of the protein. The size of the protein after the electrophoresis was ran was approxiamately 28 kilodaltons. This can be concluded by comparing the dried gel to the molecular weight standard that was printed from the internet. Purification produced a yield of .495mg. After converting the 28 kilodaltons to miligrams, the new value was 4.65*10^-17mg. The high difference of the two values was probably due to the protein from purification not being nearly as pure as the protein once electrophoresis took place. The estimated protein purity was at 50% due to two bands being present under sample 5.
Conclusions: In this lab, protein was expressed from a sample of e. coli cells which were later purified. The prepared protein was combined with a purple dye and ran through a process called electrophoresis. The heat dried product was then analyzed. The molecular weight and percent purity were estimated using a given molecular weight standard and with this information, a qualitative analysis on how well the purification went was inferred. After the experiment, a fairly pure sample of protein was produced with an estimated weight of 28 kilodaltons. The data collected after electrophoresis could be compared to the information obtained from purification in order to check for any mistakes or errors made. Although, working with proteins is often quite hands-on and innovative, it is a delicate and expensive procedure that requires time and space. Because of these, virtual drug screening can be used to speed up the drug discovery procedures while saving time and financial costs. However, protein analysis in wet lab will continue in addition to the newer methods of virtual screening used due to its head-on approach. Protein analyzing using wet-lab methods will likely be used in future VDS testing especially with newly discovered proteins while continuing research on some of the older proteins that have been in the PDB for decades.
References:
1. Structural Genomics Consortium. China Structural Genomics Consortium. Northeast Structural Genomics Consortium. Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC. Protein production and purification. Nat Methods. 2008 Feb;5 (2):135-46.
2. Bolstad, H.M.; Wood, Matthew J. An In Vivo Method for Characterization of Protein Interactions within Sulfur Trafficking Systems of E. coli.Journal of Proteome Research20109 (12), 6740-6751
Improve title
Make intro more relevant to this lab
Improve references
Images out of order
Improve captions
Flow of discussion hard to follow
Missing error analysis
Title: Successfully Preparing and Characterizing a Protein Sample
Introduction: This was a three part lab that was began the week before spring break and ended just this last week.The first part involved protein expression followed by purification and then characterization. Protein expression involves the separation of the protein from the actual bacterial cells. Purification is the process used to further isolate a target protein using different scientific process and characterization involves prepping the protein samples for identification and analysis using gel electrophoresis. Recombinant DNA and protein expression has become more commercially accessible due to the methods of production becoming more cost efficient to even the youngest of aspiring scientists particularly biologists and health care scientists.Workers in the this field of research generally refer to informative websites such as the Protein Data Bank to acquire knowledge on proteins before conducting research. Different types of proteins can be analyzed using purification methods. Some of these include human proteins, bacterial proteins, fungal proteins, protozoan proteins and other types of animal proteins. Before expression can occur, the protein that is going to be used in testing needs to be acquired. One of the main ways this is done is through cloning of the various kinds of proteins. This method allows for variants of the original protein to be created usually by adding different tags or expression hosts.
Materials & Methods: The first step of the lengthy process was protein expression. In order for this to occur, a sample of E. coli bacteria was heat-shocked and spun using a centrifuge to allow access to the protein. Colonies of this protein, pGR-22, were produced over approximately twenty four hours inside of clean agar plates. LB and ampicillin were added and the final samples were harvested in Eppendorf tubes. A pellet was formed in pellet was then saved for purification. The following week, wash buffer was added and two washes were performed, purifying the protein. A NanoDrop spectrophotmeter was used to test the concentration of the protein at different wavelenght 280nM and 540 nM. In the final part of the experiment, electrophoresis was performed followed by gel drying. Before electrophoresis took place, the six samples collected from protein purication were treated with purple dye to distinguish the proteins once they were inserted into the gel. The gel was dried out in a gel dryer for one and a half hours. The finished product was compared to a molecular weight standard retrieved from the internet to determine the molecular weight and estimated purity.
Results:
For Beer's Law, the equation A=ebc is used. If concentration, wavelenght, and absorbance are given or found, then molar absorbance can be calculated from this formula. (.211)=(38,850)(1cm)(c) so c=(5.43*10^-6 moles per liter). The concentration is then multiplied by the molality (25794.2 grams per mol) which gives .140 grams per liter. or .140 miligrams per milliliter.
Discussion: Lysozyme is used during protein expression in order to break down the cell membranes of escherichia coli.This aids in preparing for bacterial protein extraction. Benzonase was used to digest any DNA and RNA from the bacterial cells in order to reduce the viscosity of the solution. Histadine is able to bond with Nickel (Ni) which is then able to be left behind with the column due to the size of the complex being too large to filter through with the protein. Sample 2 consists of the protein after the benzonase has been added and gone through the the centrifuge. Sample 3 is stored after the Ni-Nta is added to the protein and syringed in a filter. After this step is when the imidiazole is added that begins competing with the Histadine tags and a sample of this is taken as number 4. Sample 5 contains more of the protein than number 6 due to it being the first elution and sample 6 being elution 2. These elutions were performed by adding a highly concentrated amount of imidiazole to the solution used for sample 4. Again the main difference between the wash and elution buffers is the concentration of the imidazole involved with that of the elution buffers being much strong so as to strip away more of the protein. The size of the protein after the electrophoresis was ran was approxiamately 28 kilodaltons. This can be concluded by comparing the dried gel to the molecular weight standard that was printed from the internet. Purification produced a yield of .495mg. After converting the 28 kilodaltons to miligrams, the new value was 4.65*10^-17mg. The high difference of the two values was probably due to the protein from purification not being nearly as pure as the protein once electrophoresis took place. The estimated protein purity was at 50% due to two bands being present under sample 5.
Conclusions: In this lab, protein was expressed from a sample of e. coli cells which were later purified. The prepared protein was combined with a purple dye and ran through a process called electrophoresis. The heat dried product was then analyzed. The molecular weight and percent purity were estimated using a given molecular weight standard and with this information, a qualitative analysis on how well the purification went was inferred. After the experiment, a fairly pure sample of protein was produced with an estimated weight of 28 kilodaltons. The data collected after electrophoresis could be compared to the information obtained from purification in order to check for any mistakes or errors made. Although, working with proteins is often quite hands-on and innovative, it is a delicate and expensive procedure that requires time and space. Because of these, virtual drug screening can be used to speed up the drug discovery procedures while saving time and financial costs. However, protein analysis in wet lab will continue in addition to the newer methods of virtual screening used due to its head-on approach. Protein analyzing using wet-lab methods will likely be used in future VDS testing especially with newly discovered proteins while continuing research on some of the older proteins that have been in the PDB for decades.
References:
1. Structural Genomics Consortium. China Structural Genomics Consortium. Northeast Structural Genomics Consortium. Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC.
Protein production and purification. Nat Methods. 2008 Feb;5 (2):135-46.
2. Bolstad, H.M.; Wood, Matthew J. An In Vivo Method for Characterization of Protein Interactions within Sulfur Trafficking Systems of E. coli.Journal of Proteome Research20109 (12), 6740-6751
Improve title
Intro not clear